Hematopoietic stem cells - self- renewing cells that give rise to the blood and immune systems - serve one of the most crucial roles in the body, spawning components that protect the body from illness and nourish tissues with oxygen.
These cells also may be the most promising tools to cure many life-threatening genetic diseases, such as severe anemias and immunodeficiencies, if they could be modified efficiently to contain healthy copies of missing or defective genes.
A major obstacle to the success of stem-cell gene therapy has been the limited ability to efficiently transfer genetic material into these cells.
But according to a new Hutch study, gene transfer to hematopoietic stem cells could be improved considerably using a gene-delivery system containing a portion of a feline virus.
Tested thus far only in animals, the strategy may be useful for treating human blood disorders caused by genetic defects, said Dr. Hans-Peter Kiem, lead author of the study and an investigator in the Clinical Research Division.
"Gene-therapy research has come quite a ways in the last five or six years," he said. "We have improved the gene-transfer efficiency into hematopoietic stem cells from less than one one-hundredth of a percent to about 10 percent, which is really quite significant."
Kiem and colleagues reported gene marking and expression in up to10 percent of blood cells after transplantation of genetically modified stem cells, using a gene-delivery system containing an envelope protein from a feline virus. The study appears in the Oct. 1 issue of Blood.
Other investigators on the study included Dr. Martin Goerner, now at the University of Heidelberg in Germany, Laura Peterson and Drs. Peter Horn, Peter Kurre and Rainer Storb of the Clinical Research Division, and Dr. John Rasko, now at Centenary Institute of Cancer Medicine and Cell Biology in Sidney, Australia.
Gene therapy tries to introduce healthy versions of genes into cells of tissues that lack them. The strategy requires a gene-delivery system, or vector, to transfer DNA into cells. Viruses are attractive vectors because they can be engineered to infect specific cell types and modified to reduce or eliminate their pathogenic properties. Particularly useful are retroviruses, which insert their genetic material into the chromosomes of host cells and become a permanent fixture of the cell's DNA.
Stable persistence of the genes in tissue requires gene transfer into long-lived adult stem cells that can self-renew and give rise to specialized cell types that make up a tissue. Hematopoietic stem cells are the most accessible adult stem cells, as they can be isolated from peripheral blood.
Previous studies by Kiem, Kurre and Dr. Dusty Miller of the Human Biology Division have shown that the type of viral envelope protein, the molecule that coats the virus and determines which cell type it can infect, can have a significant effect on the transfer of the virus into cells. Cells must produce specific receptor molecules on their surfaces that are recognized by viral envelope proteins, thus allowing infection to take place.
"How the virus is packaged really makes a difference on the efficiency of gene transfer, and it's important to have high-level expression of the virus receptor on the cell surface," Kiem said.
The new study compared gene-transfer efficiency of viruses with an envelope protein derived from a feline virus to viruses with an envelope protein from a gibbon-ape leukemia virus. Hematopoietic stem cells were isolated from peripheral blood and divided into portions infected with each virus. The modified stem cells were reintroduced into animals whose own blood and immune systems were destroyed by radiation.
Presence of virus in blood and immune cells was determined by looking for viral DNA and cell fluorescence, since some of the viruses were engineered to contain the marker gene for green fluorescent protein, said Kiem, whose lab has extensive expertise in gene marking and tracking technology.
"What's new and exciting in this study is that in addition to the persistence of marked cells, we see that a high number of blood cells also express the introduced gene," he said, "and we can detect green fluorescent protein in multiple cell types - T cells, granulocytes, red blood cells and platelets."
The feline-virus envelope protein also has been shown to mediate efficient gene transfer into human hematopoietic cells, which holds promise for treating blood disorders with gene therapy.
"We hope to use this method in the next year to treat patients with Fanconi anemia," Kiem said. "Currently, the only curative treatment for this disease is a stem-cell transplant. But without a related donor, the complications are serious."
Fanconi anemia is a rare genetic disorder that results in destruction of the bone marrow. Patients with the disease, caused by a mutation in one of eight different genes, are frequently diagnosed in early childhood, and afflicted children rarely reach adulthood. In addition to severe anemia, Fanconi children are susceptible to developing cancers, including acute myelogenous leukemia.
At this time, a hematopoietic stem-cell transplant is the only potential cure for the disease. While transplantation always carries risk, Fanconi patients fare much worse than other transplant candidates when a donor is mismatched or unrelated.
Kiem and other U.S. researchers hope to use gene therapy to correct the genetic defect in the hematopoietic stem cells of Fanconi anemia patients. Because a patient's own hematopoietic stem cells would be modified during this procedure, subsequent transfusion of these genetically altered cells back into the patient would pose no risk of graft-vs.-host disease, a frequent complication of unrelated transplants.
The technique also could be applied to other disorders, including diseases that impair the oxygen-carrying molecule hemoglobin or that cause immunodeficiences. University of Washington collaborators, including Dr. George Stamatoyannopoulos, professor of genetics and medicine, and Dr. Hans Ochs, professor of pediatrics, immunology and rheumatology, have active research programs in these areas.
Kiem's lab is studying whether certain types of viruses can successfully infect stem cells from other tissues, such as muscle.
"Hematopoietic stem cells are the most accessible cells," he said. "But we can also get efficient transfer into cells from other tissues. The challenge right now is to determine whether the modified cells end up in the right place. That is where gene-marking technology really becomes important."